Piezo1 and Piezo2 ion channels are essential for our senses of touch and proprioception, and the detection of lung stretch and vascular blood flow. As of today, 8 distinct human diseases have been associated with 61 single-point mutations in Piezos, many of which are not obviously related to their known physiological functions. While for most mutations their effects on Piezo function are unknown, the few mutations studied thus far distinctly affect Piezo inactivation, which is itself not understood mechanistically. The overall objective of this application is a comprehensive functional characterization of all currently-known human disease-related mutations in mechanically-activated Piezo ion channels and solving the mechanism of inactivation. Our rationale is that by determining functional effects of each point-mutation and by knowing the mechanism of Piezo inactivation we take the two first steps necessary for understanding these diseases. Our central hypothesis is that single-point mutations in Piezos that have been associated with human diseases affect membrane expression, ion permeation, or open probability, and that Piezo inactivation is determined by specific structures (residues/domains) within the C-terminal-extracellular domain (CED). The scientific premise for this hypothesis is based on the facts, that i) human patients diagnosed with colorectal polyposis, dehydrated stomatocytosis, lymphatic dysplasia, hemolytic anemia, and distal arthrogryposis, Marden-Walker syndrome, Gordon syndrome, microphthalmia are associated with mutations in Piezo1 and Piezo2, respectively, that ii) inactivation is conferred by the CED and the known main target of functional modulation of Piezos by either mutations, ligands, and voltage, and iii) our own studies showing that human disease-related point-mutations that alter inactivation kinetics profoundly change transduction of repetitive mechanical stimuli, which Piezos likely encounter during mechanical vibrations, repetitive lung stretch during breathing, or pulsating blood flow upon heart beating. Our specific aims will test the following hypotheses: Aim1: Determine the effects of 61 single-point mutations on Piezo1 and Piezo2 function; Aim2: Identification of the structures and molecular mechanism of inactivation. The proposed research is innovative, because we explore the functional consequences of 61 human Piezo1 and Piezo2 disease-related single-point mutations, nearly all of which have remained uncharacterized on a functional level, and because we will identify the mechanism of inactivation and its structural correlates, both of which are currently unknown. The significance of this study is a comprehensive biophysical analysis of functional effects of Piezo point-mutations that have been associated with human diseases of unknown mechanisms, and the mechanistic and structural exploration of inactivation as their target. This knowledge will give deep insight into the mechanisms underlying these diseases and guide strategies for further mechanistic explorations, effective diagnosis and disease treatment.